The applicant's long-term goal is to determine the cholesterol delivery mechanisms and their regulation. Cholesterol delivery to cells and tissues is a critical process in maintaining normal cell function. Specifically, regulatory roles of free cholesterol (FC) and free fatty acids (FFA) on cholesterol delivery have been of interest since they affect cell lipid metabolism at various levels; influx and efflux of lipoproteins as well as direct effects on cell receptors and lipogenic processes at genetic levels. The proposed studies will examine selective uptake (SU) as a mechanism for cell cholesterol delivery and its regulation by FC and FFA, and will allow the applicant to expand his research competition. New techniques such as fluorescent microscopy and use and generation of transgenic mice used in studies herein will provide new skills for future studies. Our previous studies demonstrated that J774 macrophages incubated with FFA increased not only LDL uptake by the LDL receptor (LDLR), but also markedly stimulated SU of cholesteryl esters (CE) from LDL. Although LDL uptake via LDLR is a primary cholesterol delivery pathway, other pathways such as SU are important in cells and tissues with little or no LDLR. Since an initial step of SU likely involves the CE transfer to the plasma membrane, the role of the plasma membrane, particularly FC and sphingolipid rich membrane rafts, as a CE acceptor will be determined by assessing transfer of (3H) cholesteryl ethers from LDL core to isolated membranes or large unilamellar vesicle (LUV) as model membranes. Moreover, since FC modulates membrane fluidity, changes in FC content in membranes on CE transfer from LDL will be examined (Aim 1). Similarly, potential roles of FFA on regulating SU will be examined by directly intercalating various FFA, or altering phospholipid content in the plasma membrane that occurs in cells incubated with FFA (Aim 2). The second step of SU, internalization of membrane CE, will be addressed by using fluorescently labeled LDL, where LDL-CE and apolipoprotein B (apoB) will be traced with fluorescent BODIPY and ALEXA, respectively. Studies will be performed to determine what cell organelles are required for CE internalization by disrupting various cell organelles with specific inhibitors. We will determine whether membrane rafts are required for CE internalization and whether FFA affect raft-mediated CE trafficking by in situ colocalization of CE and membrane rafts in the presence or absence of FFA. Also, two different SU pathways, mediated by lipoprotein lipase vs. FFA, will be compared to determine whether CE internalization by these pathways are different (Aim 3). Finally, the physiological relevance of LDL SU in tissues will be determined using mutant mice and whether dietary intakes of different fats modulate cholesterol delivery to tissues via SU from LDL and whether macrophage specific expression of LpL stimulates cholesterol delivery to extrahepatic tissues (Aim 4). Roles of SU and its regulation by FC and FFA will be delineated and results from these studies will provide further understanding on mechanisms of cholesterol delivery to cells.